1. The Field of the Invention
The present invention relates to a process for preparing a very pure, non-immunogenic, homogeneous alpha-interferon having antiviral and immunoregulatory activity, the protein itself and the use thereof.
2. The Background Art
Interferons are proteins naturally ocurring in the body which may be detected in a great variety of species. The antiviral and immunoregulatory properties inherent in them indicated at an early stage that they might be suitable for a wide variety of applications. Tests have shown that there are different classes of interferon. In addition to alpha, beta and gamma interferons, omega-interferon has recently been discovered and its structure clarified.
The high expectations placed on the interferons as an effective agent against viral diseases and cancer have already lead to trials with interferon preparations obtained from natural material, but serious side effects occurred. The preparations used in these trials, even after laborious purification, contained complex mixtures of different interferons and, in many cases, other proteins. The reason for this is that some of the interferons have subtypes differing from one another to a greater or lesser extent; thus, for example, more than 20 different types of alpha-interferon are known.
Only by producing interferons by genetic engineering has it been possible to conduct trials with pure types of interferon preparations.
These also include the recombinant alpha.sub.2 interferons used in the clinical trials (also known as alpha A). The purification of the proteins is of critical importance to the production of human proteins by microorganisms. Any contamination originating from the host organism would lead to immune defense reactions if the product were to be used in humans and these could be life threatening. However, nowadays, the removal of contaminants of this kind presents scarcely any problems and the extremely sensitive analytical methods now available will detect endotoxins in very tiny concentrations. In the field of interferon research too, methods of purification have been developed with which interferon preparations containing virtually no endotoxins can be obtained. Mention may be made, for example, of the work of Staehelin et al., J. Biol. Chem. 256, 9750 (1981).
In addition, recombinant alpha-interferons used in clinical trials are virtually free from endotoxins.
It was, therefore, all the more surprising that side effects were encountered that were severe enough to require discontinuation of interferon treatment. Some recombinant alpha-interferons were, surprisingly, found to be immunogenic; antibodies against interferon had been stimulated (Quesada et al., J. Natl. Cancer Inst. 70, No. 6, 1041-1046 (1983); Protzman et al., J. Immunol. Methods 75, 317-323 (1984)).
These antibodies may lead to serious effects if they influence the action of the interferon. This is because they act, not only on the recombinant alpha-interferon, but, as the recombinant alpha-interferon is identical to the body's own interferon, on the body's own interferon as well.
The disastrous aspect of this is that these antibodies go on acting even after the interferon treatment has ended. The antibodies may cause a deterioration during the course of the disease, weakening the body's own defenses against virus infections, and thus making the organism even more susceptible to other infections.
These effects have already been confirmed in tests on animals. Therefore, with a view to maximum safety of drug treatment, it is essential that the recombinant alpha.sub.2 interferon must be pure and virtually free from endotoxins and non-immunogenic.
The cause of the immunogenicity of the abovementioned alpha-interferons is not known. All that is clear is that endotoxic contaminants are not responsible.
The preparations of alpha-interferon that have been used for trials differ primarily in slight variations in the basic amino acids at positions 23 and 34:
______________________________________ Amino acid 23 Amino acid 34 ______________________________________ Preparation I: Lysine Histidine Preparation II: Arginine Histidine Preparation III: Arginine Arginine ______________________________________
In addition, the differences in the primary structure of the proteins used in clinical trials, it is well known that the alpha-interferons produced by genetic engineering always consist of a mixture of monomeric, shortened-molecular, reduced and oligomeric forms of interferon (see, for example, EPA 108 585, 110 302 and 118 808). Some of these forms show the same activities in vitro, but others show reduced activities and some are reputed to have possible immunogenic properties (see EPA 108 585 and 110 302).
These patent applications describe processes for separating these forms of interferon.
EPA 108 585 describes a process for separating a "slow moving monomer" and oligomers wherein the interferon probe is incubated at a temperature of 28.degree.-40.degree. C. for some time at a pH of 3 to 5.
EPA 110 302 describes a process wherein the monomer is formed from the oligomers by reduction with a redox system.
Finally, in EPA 118 808, recombinant alpha-interferon is purified with the aid of metal chelate resins from the oligomeric forms.
The interferons obtained by these methods are claimed to contain monomeric interferon in virtually quantitative form; however, there are no tests for immunogenicity reported in these documents.